Project Summary T cell exhaustion is a dysfunctional T cell state after chronic antigen exposure, and this exhausted T cells (TEX) are often developed in chronic infection or tumor microenvironment. One of the key features of TEX is increased expression of inhibitory molecules such as Cytotoxic T Lymphocyte Antigen 4 (CTLA-4) and Programmed Death 1 (PD-1). These molecules work as immune checkpoints to suppress T cell function in order to prevent pathological damage from T cell hyper-activation. However, in the tumor microenvironment, these molecules limit the cytotoxic effect of T cells to kill tumor. Blocking these immune checkpoints has been shown to achieve significant clinical benefits in multiple cancer types. Despite of these successes, the majority of the patients do not achieve long-term tumor remission. Thus, it is crucial to study the underlying molecular mechanism of T cell exhaustion development, and seek for potential combination strategies with immune checkpoint blockade (ICB) to enhance and sustain T cell response in clinic. During my doctoral research, I focus on investigating the transcriptional mechanisms of T cell exhaustion development, and how transcription factors control T cell reinvigoration after PD-1 blockade. In Specific Aim 1.1, I focused on the transcriptional mechanisms of effector T cell loss and TEX progenitor establishment during chronic antigen exposure. Using single cell RNA sequencing (sc-RNA-Seq), we found two distinct transcriptional networks in the chronic effector T cells and the TEX progenitors, and we systematically described the molecular mechanism of TEX development. In Specific Aim 1.2, I focus on establishing a T cell based in vivo CRISPR-Cas9 screening system (RetroCRISPR) to functionally study transcriptional mechanisms during T cell reinvigoration by PD-1 blockade. These studies will not only discover novel mechanisms of T cell exhaustion, but also provide high dimensional profiling or high throughput screening systems for the field to study T cell response. In Specific Aim 2, I plan to extend my RetroCRISPR system into dissecting the complexity of tumor microenvironment, and I will search for potential combination strategies with ICB to enhance T cell response. The overall goal for my current projects and future plans is to discover cell intrinsic and extrinsic mechanisms affecting T cell response, and design optimal immune therapeutic strategies for long-term tumor remission.